Abstract
The broad mite, Polyphagotarsonemus latus (Banks) (Acari: Tarsonemidae), has been a devastating pest of several important agricultural and horticultural crops. Studies on the molecular mechanisms of adaptation to extrinsic conditions, e.g. acaricide exposure and temperature stress could aid in revealing the mite’s xenobiotic metabolism and quick adaptation to rapidly changing environments, respectively. This is best studied by the gene expression patterns, which could be accomplished through real-time quantitative reverse transcription PCR (qRT-PCR). Since real-time qRT-PCR studies require the selection of one or more reference genes whose expression patterns might vary across treatments, it is indispensable to validate their expression stability. In this study, the expression stability of six candidate reference genes viz., actin, elongation factor (EF-1α), glyceraldehyde-3-phosphate-dehydrogenase (GAPDH), ribosomal protein 49 (RP49), ribosomal protein S18 (RPS18) and β-tubulin (TUB) was investigated through real-time qRT-PCR experiments. By integrating the results of geNorm, NormFinder, BestKeeper, comparative ΔCt and RefFinder algorithms, RPS18/RP49 and EF-1α/RPS18 were identified as highly suitable reference genes for acaricide treatment and temperature stress treatments, respectively. RPS18 was recognized as the most suitable reference gene for both treatments which will ensure the accuracy of target gene expressions in studies related to xenobiotic metabolism and stress tolerance in P. latus.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
Abbreviations
- Ct:
-
Cycle threshold
- EF-1α :
-
Elongation factor
- GAPDH :
-
Glyceraldehyde-3-phosphate-dehydrogenase
- RP49 :
-
Ribosomal protein 49
- RPS18 :
-
Ribosomal protein S18
- TUB :
-
β-Tubulin
- M value:
-
Expression stability value
References
Andersen CL, Jensen JL, Ørntoft TF (2004) Normalization of real-time quantitative reverse transcription-PCR data: a model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets. Cancer Res 64:5245–5250. https://doi.org/10.1158/0008-5472.CAN-04-0496
Arunkumar KM, Srinivasa N (2021) Spatial and temporal distribution of yellow mite (Polyphagotarsonemus latus) on mulberry. Mysore J Agric Sci 55:28–38
Augustine N, Thiruvengadam V, Selvapandian U, Muthugounder M (2023) Acaricide resistance among broad mite (Polyphagotarsonemus latus (Banks)) populations in Karnataka, India. Curr Sci 124:1462–1468. https://doi.org/10.18520/cs/v124/i12/1462-1468
Bustin SA, Benes V, Nolan T, Pfaffl MW (2005) Quantitative real-time RT-PCR- a perspective. J Mol Endocrinol 34:597–601. https://doi.org/10.1677/jme.1.01755
Dai TM, Lü ZC, Liu WX, Wan FH (2017) Selection and validation of reference genes for qRT-PCR analysis during biological invasions: the thermal adaptability of Bemisiatabaci MED. PLoS ONE 12:e0173821. https://doi.org/10.1371/journal.pone.0173821
de Coss-Romero M, Peña JE (1998) Relationship of broad mite (Acari: Tarsonemidae) to host phenology and injury levels in Capsicum annuum. Fla Entomol 1:515–526. https://doi.org/10.2307/3495950
de Santana MF, de Oliveira JV, Breda MO, Silva Barbosa DR, Filho AB, de Oliveira CM (2021) Host preference, acaricides effects and population growth of Polyphagotarsonemus latus (Banks) (Acari: Tarsonemidae) on white and coloured cotton cultivars. Pest Manag Sci 77:217–223. https://doi.org/10.1002/ps.6010
Johnson G, Nolan T, Bustin SA (2013) Real-time quantitative PCR, pathogen detection and MIQE. In: Wilks M (ed) PCR detection of microbial pathogens. Methods in molecular biology. Humana Press, Totowa, NJ, pp 89–90
Kwon DH, Park JH, Lee SH (2013) Screening of lethal genes for feeding RNAi by leaf disc-mediated systematic delivery of dsRNA in Tetranychus urticae. Pestic Biochem Physiol 105:69–75. https://doi.org/10.1016/j.pestbp.2012.12.001
Lee J, Kim YH, Kim K, Kim D, Lee SH, Kim S (2022) Selection of stable reference genes for quantitative real-time PCR in the Varroa mite Varroa destructor. Arch Insect Biochem Physiol 110:e21905. https://doi.org/10.1002/arch.21905
Linghua X, Baosheng Z, Noland Jeffery E, Yongping H, Xuguo Z (2015) The coming of RNA-based pest controls. J Plant Prot 4:673–690. https://doi.org/10.13802/j.cnki.zwbhxb.2015.05.001
Liu J, Jiang Z, Feng K, Lu W, Wen X, Sun J, Li J, Liu J, He L (2021) Transcriptome analysis revealed that multiple genes were related to the cyflumetofen resistance of Tetranychus cinnabarinus (Boisduval). Pestic Biochem Physiol 173:104799. https://doi.org/10.1016/j.pestbp.2021.104799
Lourenço AP, Mackert A, dos Santos CA, Simões ZL (2008) Validation of reference genes for gene expression studies in the honey bee, Apis mellifera, by quantitative real-time RT-PCR. Apidologie 39:372–385. https://doi.org/10.1051/apido:2008015
Morales MA, Mendoza BM, Lavine LC, Lavine MD, Walsh DB, Zhu F (2016) Selection of reference genes for expression studies of xenobiotic adaptation in Tetranychus urticae. Int J Biol Sci 12:1129. https://doi.org/10.7150/ijbs.16319
Nasrin M, Amin MR, Miah MR, Akanda AM, Miah MG, Kwon O, Suh SJ (2021) Occurrence and severity of mite Polyphagotarsonemus latus (Tarsonemidae) on chili plants: Analysis of pest-weather and host plant characteristics. Entomol Res 51:273–281. https://doi.org/10.1111/1748-5967.12491
Niu JZ, Dou W, Ding TB, Yang LH, Shen GM, Wang JJ (2012) Evaluation of suitable reference genes for quantitative RT-PCR during development and abiotic stress in Panonychus citri (McGregor) (Acari: Tetranychidae). Mol Biol Rep 39:5841–5849. https://doi.org/10.1007/s11033-011-1394-x
Niu D, Zhao Y, Gong X, Zhang W, Yang R, Hu L, Xiong G, Ding S (2019) Screening of reference genes and quantitative real-time PCR detection and verification in Dermatophagoidesfarinae under temperature stress. Exp Parasitol 206:107754. https://doi.org/10.1016/j.exppara.2019.107754
Perrin BJ, Ervasti JM (2010) The actin gene family: function follows isoform. Cytoskeleton 67:630–634. https://doi.org/10.1002/cm.20475
Pfaffl MW, Tichopad A, Prgomet C, Neuvians TP (2004) Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: bestkeeper-excel-based tool using pair-wise correlations. Biotechnol Lett 26:509–515. https://doi.org/10.1023/B:BILE.0000019559.84305.47
Price DR, Gatehouse JA (2008) RNAi-mediated crop protection against insects. Trends Biotechnol 26:393–400. https://doi.org/10.1016/j.tibtech.2008.04.004
Rai AB, Satpathy S, Gracy GR, Swamy TMS, Mathura R (2007) Yellow mite Polyphagotarsonemus latus (Banks) menace in chilli crop. Veg Sci 34:1–13
Rodríguez IV, Mesa-Cobo NC, Valencia MO, Ossa J (2017) Population parameters and damage of Polyphagotarsonemus latus (Acari: Tarsonemidae) in Valencia orange (Citrus sinensis [L.] Osbeck) crop. Acta Agron 66:633–640. https://doi.org/10.15446/acag.v66n4.59922
Shakeel M, Rodriguez A, Tahir UB, Jin F (2018) Gene expression studies of reference genes for quantitative real-time PCR: an overview in insects. Biotechnol Lett 40:227–236. https://doi.org/10.1007/s10529-017-2465-4
Silver N, Best S, Jiang J, Thein SL (2006) Selection of housekeeping genes for gene expression studies in human reticulocytes using real-time PCR. BMC Mol Biol 7:1–9. https://doi.org/10.1186/1471-2199-7-33
Sousa VC, Zélé F, Rodrigues LR, Godinho DP, de La Masselière MC, Magalhães S (2019) Rapid host-plant adaptation in the herbivorous spider mite Tetranychus urticae occurs at low cost. Curr Opin Insect Sci 36:82–89. https://doi.org/10.1016/j.cois.2019.08.006
Srinivasan MR, Natarajan N, Palaniswamy S (2003) Evaluation of buprofezin 25 SC and fenpyroximate 5 SC against chilli mite, Polyphagotarsonemus latus (Banks). Indian J Pl Prot 31:116–117
Stahlberg A, Hakansson J, Xian X, Semb H, Kubista M (2004) Properties of the reverse transcription reaction in mRNA quantification. Clin Chem 50:509–515. https://doi.org/10.1373/clinchem.2003.026161
Sullivan-Gunn M, Hinch E, Vaughan V, Lewandowski P (2011) Choosing a stable housekeeping gene and protein is essential in generating valid gene and protein expression results. Br J Cancer 104:1055. https://doi.org/10.1038/bjc.2011.35
Sun W, Jin Y, He L, Lu WC, Li M (2010) Suitable reference gene selection for different strains and developmental stages of the carmine spider mite, Tetranychus cinnabarinus, using quantitative real-time PCR. J Insect Sci 10:201–205. https://doi.org/10.1673/031.010.20801
Suzuki T, Nunes MA, España MU, Namin HH, Jin P, Bensoussan N, Zhurov V, Rahman T, De Clercq R, Hilson P, Grbic V (2017) RNAi-based reverse genetics in the chelicerate model Tetranychus urticae: a comparative analysis of five methods for gene silencing. PLoS ONE 12:e0180654. https://doi.org/10.1371/journal.pone.0180654
Thellin O, Zorzi W, Lakaye B, De Borman B, Coumans B, Hennen G, Grisar T, Igout A, Heinen E (1999) Housekeeping genes as internal standards: use and limits. J Biotechnol 75:291–295. https://doi.org/10.1016/S0168-1656(99)00163-7
Van Hiel MB, Van Wielendaele P, Temmerman L, Van Soest S, Vuerinckx K, Huybrechts R, Broeck JV, Simonet G (2009) Identification and validation of housekeeping genes in brains of the desert locust Schistocerca gregaria under different developmental conditions. BMC Mol Biol 10:1–10. https://doi.org/10.1186/1471-2199-10-56
Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, Speleman F (2002) Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 3:1–12. https://doi.org/10.1186/gb-2002-3-7-research0034
Vlogiannitis S, Mavridis K, Dermauw W, Snoeck S, Katsavou E, Morou E, Harizanis P, Swevers L, Hemingway J, Feyereisen R, Van Leeuwen T (2021) Reduced proinsecticide activation by cytochrome P450 confers coumaphos resistance in the major bee parasite Varroa destructor. Proc Natl Acad Sci 118:e2020380118. https://doi.org/10.1073/pnas.2020380118
Wang C, Xu X, Huang Y, Yu H, Li H, Wan Q, Pan B (2020) Transcription profiling and characterization of Dermanyssusgallinae cytochrome P450 genes involved in beta-cypermethrin resistance. Vet Parasitol 283:109155. https://doi.org/10.1016/j.vetpar.2020.109155
Xie F, Xiao P, Chen D, Xu L, Zhang B (2012) miRDeepFinder: a miRNA analysis tool for deep sequencing of plant small RNAs. Plant Mol Biol 80:75–84. https://doi.org/10.1007/s11103-012-9885-2
Yang C, Pan H, Liu Y, Zhou X (2015) Stably expressed housekeeping genes across developmental stages in the two-spotted spider mite, Tetranychus Urticae. PLoS ONE 10:e0120833. https://doi.org/10.1371/journal.pone.0120833
Yang J, Gao Y, Liu Z, Lu J, Zhang Y, Zhang P, Fan J, Zhou X, Fan R (2019) Selection of reference genes for RT-qPCR analysis under intrinsic conditions in the hawthorn spider mite, Amphitetranychus viennensis (Acarina: Tetranychidae). Front Physiol 10:1427. https://doi.org/10.3389/fphys.2019.01427
Yang J, Zhang Y, Zhao J, Gao Y, Liu Z, Zhang P, Fan J, Zhou X, Fan R (2020) Selection of reference genes for RT-qPCR analysis under extrinsic conditions in the hawthorn spider mite Amphitetranychus Viennensis. Front Physiol 11:378. https://doi.org/10.3389/fphys.2020.00378
Zhang Y, Zhang Z, Ren M, Liu X, Zhou X, Yang J (2022) Selection of reference genes for RT-qPCR analysis in the hawthorn spider mite, Amphitetranychus viennensis (Acarina: Tetranychidae), under acaricide treatments. J Econ Entomol 115:662–670. https://doi.org/10.1093/jee/toac019
Acknowledgements
The authors extend gratitude to the Indian Council of Agricultural Research (ICAR) for funding under the Consortium Research Platform (CRP) on Genomics project. The first author profusely thanks the Department of Science & Technology, Government of India for the award of the DST- INSPIRE fellowship (2019/IF190096). The facilities provided by the Director, ICAR- National Bureau of Agricultural Insect Resources are thankfully acknowledged.
Funding
This study was supported by the Indian Council of Agricultural Research (ICAR)- Consortium Research Platform (CRP) on Genomics project and the Department of Science & Technology, Government of India through DST- INSPIRE fellowship (2019/IF190096).
Author information
Authors and Affiliations
Contributions
All authors contributed to the study conception and design. NA conducted the experiment, performed data analysis and prepared the original draft. US analyzed the transcriptome data, selected the primer sequences and aided in primer designing. MM contributed to framing of the study, resources supply, data curation and draft corrections.
Corresponding author
Ethics declarations
Conflict of interest
The authors declare that there is no potential conflict of interest.
Ethical approval
This article does not contain any studies with human participants or animals performed by any of the authors.
Additional information
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary Information
Below is the link to the electronic supplementary material.
Rights and permissions
Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.
About this article
Cite this article
Augustine, N., Selvapandian, U., Venkatesan, T. et al. Evaluation of reference genes for expression studies in the broad mite, Polyphagotarsonemus latus (Acari: Tarsonemidae). Appl Entomol Zool 59, 31–40 (2024). https://doi.org/10.1007/s13355-023-00848-3
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s13355-023-00848-3